Comprehensive pan-cancer analysis of cfDNA methylation marks in tumors reveals complex epigenetic regulatory circuits and diagnostic biomarkers

Abstract

DNA methylation is an extensively studied, stable, and fundamental epigenetic alteration in most cancer types1. Single-base-pair resolution analyses of DNA methylation is currently feasible2. Analysis of DNA methylation, in liquid biopsies hold practice-changing potentials3-6. Despite undeniable progress, clinical translation lags behind, mainly due to: 1) Challenges associated with DNA methylation analysis. 2) Fragmentation of circulating cell-free DNA (ccfDNA), worsened by bisulfite treatment. 3) Lack of clinical validation for reported ccfDNA methylation markers. 4) Limited functional characterization of ccfDNA methylation markers in tumors7. We addressed these challenges by creating a comprehensive pan-cancer cfDNA methylation resource, utilizing pools comprising over 140 patient samples and assess the utility of this resource in over 500 patient plasma and tissue samples spanning around 15 cancer entities with different clinical phenotypes and treatment approaches. Furthermore, we developed a pan-cancer enzymatic digital PCR approach and optimized entity-specific assays for ccfDNA methylation scoring. We demonstrated that this resource can profile methylation in unexplored entities, and ccfDNA methylation patterns align with those in tumor samples. Additionally, we unveiled unconventional epigenetic regulation by methylated DNA-binding transcription factors, with tissue- and context-specific and dosage-dependent activities. This work provides a reference resource for identifying minimally invasive epigenetic markers and opens avenues for characterizing methylated DNA-binding transcription factors.